Effects of Polystyrene Microplastics on Bone-related Protein Expression, Mineralization Capacity, and Mitochondrial Function in Osteoblast-like Cells (mg-63)

Introdução: Microplastics (MPs) were recently evidenced in the human skeleton. This study aimed to evaluate the effects of polystyrene (PS-MPs) on bone-related protein expression, mineralization, and bioenergetic processes. Métodos: Osteoblast-like cells (MG-63) were exposed to fluorescent PS-MPs at 5-50 µg/mL. Internalization of PS-MPs was analyzed by confocal microscopy. Osteoprotegerin, osteocalcin, osteopontin, and DKK1 levels in cell lysates/supernatants were measured using a MilliplexTM human bone panel. Cell mineralization was evaluated through alkaline phosphatase activity (p-nitrophenyl-phosphate [pNPP] hydrolysis) and calcium deposition (o-cresolphthalein complexone assay). Mitochondrial function was assessed by monitoring oxygen consumption using high-resolution respirometry. Glucose consumption and lactate production were measured using colorimetric assays. Resultados: MG-63 internalized PS-MPs into the cytoplasm. Compared to the controls, cells exposed to PS-MPs at 10 and 25 µg/mL showed significantly reduced levels of osteocalcin (137 vs. 109 pg/mL, p=0.04; and 137 vs. 100 pg/mL, p=0.03) and osteoprotegerin (8719 vs. 7402 pg/mL, p=0.04; 8719 vs. 6950 pg/mL, p=0.01) expression. DKK1 concentration was also reduced following PS exposure at 25 µg/mL (10431 vs. 7789 pg/mL, p=0.04). Similar PNP production and calcium deposition were observed. No significant differences were observed in oxygen consumption between control and PS-MPs-treated cells, regardless of the respiratory state. The median O2 flux at the H+ leak state (i.e., oligomycin present) (pmol O2/s*106 cells) for control, 5 µg/mL, and 50 µg/mL were 38.7 (33.1-45.3), 38.6 (32.8-60.4), and 30.4 (25.4-59.7). The median maximum O2 flux was 93.9 (39.2-140) for control, 105 (56.3-153.0) for the 5 µg/mL exposure, and 101 (78.4-145) for the 50 µg/mL. Glucose concentrations (mg/dL) in the cell culture medium at 48 hours (initial concentration: 100 mg/mL) were 40.4±21.4 for the control, 49.9±20.2 for the 5 µg/mL group, and 45.2±19 for the 50 µg/mL group (p>0.05). Lactate concentrations were significantly higher at 48 hours (p<0.001) for all conditions compared to their 24-hour baseline, without differences between groups. Discussão e Conclusões: PS-MPs altered the expression of key bone-related proteins without affecting calcium deposition induced by alkaline phosphatase activity. No significant differences were found in mitochondrial respiration, glucose consumption, or lactate production between the experimental conditions.